Chromatin position effects assayed by thousands of reporters integrated in parallel.

Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.

Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks.

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.

Origin of Enhancement of Orbital Magnetic Moment in SiO2-Coated Fe3O4 Nanocomposites Studied by X-ray Magnetic Circular Dichroism.

In this study, magnetic Fe3O4 nanoparticles (NPs) were dispersed uniformly by varying the thickness of the SiO2 coating, and their electronic and magnetic properties were investigated. X-ray diffraction confirmed the structural configuration of monophase inverse-spinel Fe3O4 NPs in nanometer size. Scanning electron microscopy revealed the formation of proper nonporous crystallite particles with a clear core-shell structure with silica on the surface of Fe3O4 NPs. The absorption mechanism studied through the zeta potential indicates that SiO2-coated Fe3O4 nanocomposites (SiO2@Fe3O4 NCs) possess electrostatic interactions to control their agglomeration in stabilizing suspensions by providing a protective shield of amorphous SiO2 on the oxide surface. High-resolution transmission electron microscopy images demonstrate a spherical morphology having an average grain diameter of ∼11-17 nm with increasing thickness of SiO2 coating with the addition of a quantitative presence and proportion of elements determined through elemental mapping and electron energy loss spectroscopy studies. Synchrotron-based element-specific soft X-ray absorption spectroscopy and X-ray magnetic circular dichroism (XMCD) techniques have been involved in the bulk-sensitive total fluorescence yield mode to understand the origin of magnetization in SiO2@Fe3O4 NCs. The magnetization hysteresis of Fe3O4 was determined by XMCD. At room temperature, the magnetic coercivity (Hc) is as high as 1 T, which is about 2 times more than the value of the thin film and about 5 times more pronounced than that of NPs. For noninteracting single-domain NPs with the Hc spread from 1 to 3 T, the Stoner-Wohlfarth model provided an intriguing explanation for the hysteresis curve. These curves determine the different components of Fe oxides present in the samples that derive the remnant magnetization involved in each oxidation state of Fe and clarify which Fe component is responsible for the resultant magnetism and magnetocrystalline anisotropy based on noninteracting single-domain particles.

TRIM28 is an Epigenetic Barrier to Induced Pluripotent Stem Cell Reprogramming.

Since the discovery of induced pluripotent stem cells there has been intense interest in understanding the mechanisms that allow a somatic cell to be reprogrammed back to a pluripotent state. Several groups have studied the alterations in gene expression that occur as somatic cells modify their genome to that of an embryonic stem cell. Underpinning many of the gene expression changes are modifications to the epigenetic profile of the associated chromatin. We have used a large-scale shRNA screen to identify epigenetic modifiers that act as barriers to reprogramming. We have uncovered an important role for TRIM28 in cells resisting transition between somatic and pluripotent states. TRIM28 achieves this by maintaining the H3K9me3 repressed state and keeping endogenous retroviruses (ERVs) silenced. We propose that knockdown of TRIM28 during reprogramming results in more plastic H3K9me3 domains, dysregulation of genes nearby H3K9me3 marks, and up regulation of ERVs, thus facilitating the transition through reprogramming. Stem Cells 2017;35:147-157.

Chromatin landscapes of retroviral and transposon integration profiles.

The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of ~ 120,000 to ~ 180,000 unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV). We analyzed ~ 80 (epi)genomic features to generate bias maps at both local and genome-wide scales. MMTV showed a remarkably uniform distribution of integrations across the genome. More distinct preferences were observed for the two transposons, with PB showing remarkable resemblance to bias profiles of the Murine Leukemia Virus. Furthermore, we present a model where target site selection is directed at multiple scales. At a large scale, target site selection is similar across systems, and defined by domain-oriented features, namely expression of proximal genes, proximity to CpG islands and to genic features, chromatin compaction and replication timing. Notable differences between the systems are mainly observed at smaller scales, and are directed by a diverse range of features. To study the effect of these biases on integration sites occupied under selective pressure, we turned to insertional mutagenesis (IM) screens. In IM screens, putative cancer genes are identified by finding frequently targeted genomic regions, or Common Integration Sites (CISs). Within three recently completed IM screens, we identified 7%-33% putative false positive CISs, which are likely not the result of the oncogenic selection process. Moreover, results indicate that PB, compared to SB, is more suited to tag oncogenes.

TRIP through the chromatin: a high throughput exploration of enhancer regulatory landscapes.

Enhancers are regulatory elements that promote gene expression in a spatio-temporal way and are involved in a wide range of developmental and disease processes. Both the identification and subsequent functional dissection of enhancers are key steps in understanding these processes. Several high-throughput approaches were recently developed for these purposes; however, in almost all cases enhancers are being tested outside their native chromatin context. Until recently, the analysis of enhancer activities at their native genomic locations was low throughput, laborious and time-consuming. Here, we discuss the potential of a powerful approach, TRIP, to study the functioning of enhancers in their native chromatin environments by introducing sensor constructs directly in the genome. TRIP allows for simultaneously analyzing the quantitative readout of numerous sensor constructs integrated at random locations in the genome. The high-throughput and flexible nature of TRIP opens up potential to study different aspects of enhancer biology at an unprecedented level.

Comparison of microcoils and polyvinyl alcohol particles in selective microcatheter angioembolization of non variceal acute gastrointestinal hemorrhage.

OBJECTIVES: To compare the efficacy of polyvinyl alcohol (PVA) particles with microcoils in angiembolisation of non variceal acute gastrointestinal haemorrhage. METHODS: This is a retrospective cross-sectional study of patients who underwent transcatheter angioembolization from January, 1995 to December, 2013 at Aga Khan University Hospital, Karachi. Patients were divided into two groups on basis of use of either microcoils or PVA particles and compared in terms of technical success, clinical success, re-bleeding and ischemic complication rates. Chi (χ(2)) square and Fisher's exact tests were applied and a P-value of less than 0.05 was considered statistically significant. RESULTS: Fifty seven patients underwent angioembolization. Microcoil and PVA particles embolization was performed in 63% (36/57) and 35% (20/57) cases respectively. Technical success was achieved in all cases (100%). Clinical success rate was higher in microcoils group (92%) than PVA particles group (75%) with statistically significant P value (p=0.048). Ischemic complication was seen in one case (3%) in the microcoil group, while no such complications were seen in the PVA particles group. CONCLUSION: In angioembolization of non variceal acute gastrointestinal haemorrhage microcoils are better than Polyvinyl alcohol particles with higher clinical success and lower re-bleed rates.

Nucleotide composition-linked divergence of vertebrate core promoter architecture.

Transcription initiation involves the recruitment of basal transcription factors to the core promoter. A variety of core promoter elements exists; however for most of these motifs, the distribution across species is unknown. Here we report on the comparison of human and amphibian promoter sequences. We have used oligo-capping in combination with deep sequencing to determine transcription start sites in Xenopus tropicalis. To systematically predict regulatory elements, we have developed a de novo motif finding pipeline using an ensemble of computational tools. A comprehensive comparison of human and amphibian promoter sequences revealed both similarities and differences in core promoter architecture. Some of the differences stem from a highly divergent nucleotide composition of Xenopus and human promoters. Whereas the distribution of some core promoter motifs is conserved independently of species-specific nucleotide bias, the frequency of another class of motifs correlates with the single nucleotide frequencies. This class includes the well-known TATA box and SP1 motifs, which are more abundant in Xenopus and human promoters, respectively. While these motifs are enriched above the local nucleotide background in both organisms, their frequency varies in step with this background. These differences are likely adaptive as these motifs can recruit TFIID to either CpG island or sharply initiating promoters. Our results highlight both the conserved and diverged aspects of vertebrate transcription, most notably showing co-opted motif usage to recruit the transcriptional machinery to promoters with diverging nucleotide composition. This shows how sweeping changes in nucleotide composition are compatible with highly conserved mechanisms of transcription initiation.

Coherent storage of photoexcited triplet states using 29Si nuclear spins in silicon.

Pulsed electron paramagnetic resonance spectroscopy of the photoexcited, metastable triplet state of the oxygen-vacancy center in silicon reveals that the lifetime of the m(s)=±1 sublevels differs significantly from that of the m(s)=0 state. We exploit this significant difference in decay rates to the ground singlet state to achieve nearly ~100% electron-spin polarization within the triplet. We further demonstrate the transfer of a coherent state of the triplet electron spin to, and from, a hyperfine-coupled, nearest-neighbor (29)Si nuclear spin. We measure the coherence time of the (29)Si nuclear spin employed in this operation and find it to be unaffected by the presence of the triplet electron spin and equal to the bulk value measured by nuclear magnetic resonance.

Observer variation in MRI evaluation of patients with suspected lumbar disc herniation and nerve root compression: comparison of neuroradiologist and neurosurgeon's interpretations.

OBJECTIVE: To analyse inter-observer variation between a neuroradiologist and neurosurgeon in the MRI diagnosis of lumbar nerve root compression. Although lumbar MFI is primarily analyzed and reported by a radiologist, neurosurgeons often analyse it independently as they have sufficient clinical background as well as radiological expertise to diagnose most spinal pathologies on Magnetic Resonance Imaging (MRI). METHODS: Retrospective analysis was carried out for images of 54 patients who underwent MRI between March and July 2010 of lumbar spine with suspected lumbar disc herniation and nerve root compression, at Aga Khan Hospital, Karachi, Pakistan. One fellowship trained neuroradiologist and one neurosurgeon evaluated the images on PACS system separately. Both observers were unaware of the patient's clinical history and each other's findings. Lumbar discs at L3-L4, L4-L5 and L5-S1 levels were evaluated by both observers for disc disease and nerve compression. Findings were recorded on a proforma and analysed with SPPS Version 16. RESULTS: Total 162 lumbar discs were studied by both readers in 54 patients. Excellent inter-observer agreement was seen for the presence or absence of nerve root compression (Percentage agreement = 88.89%; k = 0.774; p = 0.737). For disc bulge, inter-observer agreement was fair but statistically insignificant (Percentage agreement = 72.84%; k = 0.414; p = 0.132). In case of disc herniation, although inter-observer agreement was fair, but the difference was statistically significant (Percentage agreement = 84.57%; k = 0.511; p = 0.002). CONCLUSION: Inter-observer agreement between neuroradiologist and neurosurgeon in diagnosing nerve root compression due to lumbar disc disease was excellent. Agreement regarding disc bulge and herniation was fair.

Diagnostic accuracy of magnetic resonance cholangio-pancreatography in evaluation of obstructive jaundice.

OBJECTIVE: To determine the diagnostic accuracy of magnetic resonance cholangio-pancreatography in the evaluation of obstructive jaundice taking endoscopic retrograde cholangio-pancreatography (ERCP) as the gold standard. METHODS: The cross-sectional study was conducted at the Radiology Department of the Sindh Institute of Urology and Transplantation from August, 2009, to February, 2010. It comprised 77 patients (36 males and 41 females) with clinically obstructive jaundice referred for magnetic resonance cholangio-pancreatography evaluation. The findings were compared with the gold standard. Data was analysed using SPSS version 15. RESULTS: The sensitivity of magnetic resonance cholangio-pancreatography for obstructive jaundice was 97%; specificity was 75% and accuracy was 80%, while positive predictive value (PPV) and negative predictive value (NPV) were 99% and 60% respectively. CONCLUSION: Magnetic resonance cholangio-pancreatography is a relatively quick, accurate and non-invasive imaging modality for the assessment of obstructive jaundice in ruling out potentially correctable underlying causes and reducing unnecessary invasive interventions.

The significance of eighteen rice genotypes on arsenic accumulation, physiological response and potential health risk.

Rice is an important food crop that is susceptible to arsenic (As) contamination under paddy soil conditions depending on As uptake characteristics of the rice genotypes. Here we unveiled the significance of eighteen (fine and coarse) rice genotypes against As accumulation/tolerance, morphological and physiological response, and antioxidant enzymes-enabled defense pathways. Arsenic significantly affected rice plant morphological and physiological attributes, with relatively more impacts on fine compared to coarse genotypes. Grain, shoot, and root As uptake were lower in fine genotypes (0.002, 0.020, and 0.032 mg pot-1 DW, respectively) than that of coarse (0.031, 0.60, and 1.2 mg pot-1 DW, respectively). Various biochemical (pigment contents, hydrogen peroxide, lipid peroxidation) and defense (antioxidant enzymes) plant parameters indicated that the fine genotypes, notably Kainat and Basmati-385, possessed the highest As tolerance. Arsenic-induced risk indices exhibited greater hazard quotient (up to 1.47) and carcinogenic risk (up to 0.0066) for coarse genotypes compared to the fine ones, with the greatest risk for KSK-282. This study elaborates the pivotal role of genotypic variation among rice plants in As accumulation, which is crucial for mitigating the associated human health risk. Further research is required on molecular aspects, e.g., genetic sequencing, to examine rice genotypes variation in defense mechanisms to As contamination.

Ultrasound biosafety during pregnancy: what do operators know in the developing world?: national survey findings from pakistan.

OBJECTIVES: The purpose of this study was to evaluate the knowledge and attitudes of ultrasound users toward safety aspects during routine obstetric examinations. METHODS: A self-administered questionnaire was given to all registered participants of the 10th Annual International Radiological Conference in Karachi, Pakistan. RESULTS: A total of 306 participants completed the questionnaire (response rate, 70%), including 170 consultant radiologists (55%), 90 radiology residents (30%), 31 sonographers (10%), and 15 others (5%). The mean sonography experience of the end users ± SD was 5.1 ± 5 years. Around half of the participants (45.5%) reported that there should be limitations on the use of ultrasound during low-risk pregnancy. Overall, safety knowledge was inadequate among the participants. However, those who were concerned about limitations on the use of ultrasound during pregnancy were 2.5 times more likely to be familiar with the mechanical index (P < .001) and 1.8 times more likely to be familiar with the thermal index (P < .001). CONCLUSIONS: Increased training for professionals on safety issues of ultrasound procedures during pregnancy is needed. Furthermore, the concept of the absolute safety of ultrasound procedures without knowledge of safety parameters such as the mechanical and thermal indices needs to be changed, and more clarity is required when defining parameters under which the procedures can be safely performed.

Acute vertebral compression fracture: differentiation of malignant and benign causes by diffusion weighted magnetic resonance imaging.

OBJECTIVE: To evaluate the sensitivity, specificity and accuracy of diffusion weighted (DWI) magnetic resonance imaging (MRI) in the diagnosis and differentiation between benign (osteoporotic/infectious) and malignant vertebral compression fractures in comparison with histology findings and clinical follow up. METHODS: The study was conducted at the Radiology Department, Aga Khan University Hospital (AKUH) Karachi. It was a one year cross-sectional study from 01/01/2009 to 01/01/2010. Forty patients with sixty three vertebral compression fractures were included. Diffusion-weighted sequences and apparent diffusion coefficient (ADC) images on a 1.5 T MR scanner were obtained in all patients to identify the vertebral compression fracture along with benign and malignant causes. Imaging findings were compared with histopathologic results and clinical follow-up. RESULTS: Diffusion-weighted MR imaging found to have, 92% sensitivity, 90% specificity and accuracy of 85% in differentiation of benign and malignant vertebral compression fracture while PPV and NPV were 78 % and 90% respectively. CONCLUSION: Diffusion weighted magnetic resonance imaging offers a safe, accurate and non invasive modality to differentiate between the benign and malignant vertebral compression fracture.

Focused abdominal CT scan for acute appendicitis in children: can it help in need?

OBJECTIVE: To evaluate the focused abdominal CT scan [FACT] in clinically equivocal cases of acute appendicitis in paediatric population. METHODS: A cross-sectional study was conducted at the Radiology Department of Aga Khan Hospital, from August 2007 to November 2008. A total of 84 patients (42 males & 42 females) with clinically equivocal signs and symptoms of acute appendicitis referred to radiology department for CT evaluation were studied. CT findings were compared with histopathology and clinical follow-up. RESULTS: The sensitivity of focused CT for acute appendicitis was 91%; specificity was 69% and accuracy of 76% while PPV and NPV were 58%, 94% respectively. CONCLUSION: Focused unenhanced CT is a quick, accurate and non invasive modality for the assessment of clinically equivocal cases of acute appendicitis for ruling out patients and reducing negative appendectomies.

Accuracy of computed tomography in diagnosing malignancy in solitary pulmonary lesions.

OBJECTIVE: To evaluate accuracy of Computed Tomography (CT) for diagnosing malignancy in solitary pulmonary lesions (SPLs). METHODS: A prospective cross-sectional study was conducted from 20-01-2007 to 30-06-2008 at the Radiology department, Aga Khan University Hospital (AKUH) Karachi. Fifty-three patients with solitary pulmonary lesions (SPLs) seen in prior chest x-rays or chest CT scans were referred to radiology department for CT guided biopsy. CT scan was performed for each patient prior to biopsy and CT evaluation of the SPLs was performed followed by CT guided Biopsy. Histopathological diagnosis of the lesion was taken as the gold standard. RESULTS: CT was found to be 100% sensitive, 30% specific and 87% accurate for diagnosing malignancy in solitary pulmonary lesions while PPV and NPV were 86% and 100% respectively. CONCLUSION: CT scan is highly sensitive yet non-specific and cannot be used as the definitive diagnostic modality for diagnosing malignancy in solitary pulmonary lesions.

Imaging non-collinear antiferromagnetic textures via single spin relaxometry.

Antiferromagnetic materials are promising platforms for next-generation spintronics owing to their fast dynamics and high robustness against parasitic magnetic fields. However, nanoscale imaging of the magnetic order in such materials with zero net magnetization remains a major experimental challenge. Here we show that non-collinear antiferromagnetic spin textures can be imaged by probing the magnetic noise they locally produce via thermal populations of magnons. To this end, we perform nanoscale, all-optical relaxometry with a scanning quantum sensor based on a single nitrogen-vacancy (NV) defect in diamond. Magnetic noise is detected through an increase of the spin relaxation rate of the NV defect, which results in an overall reduction of its photoluminescence signal under continuous laser illumination. As a proof-of-concept, the efficiency of the method is demonstrated by imaging various spin textures in synthetic antiferromagnets, including domain walls, spin spirals and antiferromagnetic skyrmions. This imaging procedure could be extended to a large class of intrinsic antiferromagnets and opens up new opportunities for studying the physics of localized spin wave modes for magnonics.

TBP2 is a substitute for TBP in Xenopus oocyte transcription.

BACKGROUND: TATA-box-binding protein 2 (TBP2/TRF3) is a vertebrate-specific paralog of TBP that shares with TBP a highly conserved carboxy-terminal domain and the ability to bind the TATA box. TBP2 is highly expressed in oocytes whereas TBP is more abundant in embryos. RESULTS: We find that TBP2 is proteolytically degraded upon meiotic maturation; after germinal vesicle breakdown relatively low levels of TBP2 expression persist. Furthermore, TBP2 localizes to the transcriptionally active loops of lampbrush chromosomes and is recruited to a number of injected promoters in oocyte nuclei. Using an altered binding specificity mutant reporter system we show that TBP2 promotes RNA polymerase II transcription in vivo. Intriguingly, TBP, which in oocytes is undetectable at the protein level, can functionally replace TBP2 when ectopically expressed in oocytes, showing that switching of initiation factors can be driven by changes in their expression. Proteolytic degradation of TBP2 is not required for repression of transcription during meiotic maturation, suggesting a redundant role in this repression or a role in initiation factor switching between oocytes and embryos. CONCLUSION: The expression and transcriptional activity of TBP2 in oocytes show that TBP2 is the predominant initiation factor in oocytes, which is substituted by TBP on a subset of promoters in embryos as a result of proteolytic degradation of TBP2 during meiotic maturation.

Magnetic resonance imaging findings in patients with schizophrenia.

OBJECTIVE: To determine structural abnormalities in the brain of patients with schizophrenia by Magnetic Resonance Imaging (MRI). STUDY DESIGN: Comparative study. PLACE AND DURATION OF STUDY: The Departments of Radiology and Psychiatry, the Aga Khan University Hospital, Karachi, from January 2007 to June 2008. METHODOLOGY: Thirty-three cases of schizophrenia (n=33) and thirty-three age-matched controls, (n=33) were enrolled for this study. Screening Magnetic Resonance Imaging (MRI) of brain was done in order to see structural changes in brain matter. Findings were compared among groups using chi-square and Fisher's exact test with significance at p < 0.05. RESULTS: Among the total of 66 (n=66) MRI films studied for brain abnormalities, brain atrophy, presence of septum pellucidum and enlarged Virchow-Robins spaces were significantly associated with schizophrenia (p < 0.001). There was no significant difference between cases and controls for ventricular dilatation (p=0.5). Sinusitis was mostly associated with controls and well correlated with their symptoms (p < 0.001). CONCLUSION: Brain atrophy was the most commonly seen brain change in the studied sample of patients with schizophrenia. MRI brain can be used to identify structural abnormalities in patients with schizophrenia.

Birth weight estimation--a sonographic model for Pakistani population.

OBJECTIVE: To develop a sonographic birth weight estimation model for Pakistani population and to validate the published models in the same population. METHODS: Data was collected for pregnant women who presented to Radiology Department of Aga Khan University Hospital Karachi from January 2007 to July 2008 and had undergone ultrasound estimation of foetal weight within 4 days prior to a term delivery (37-42 weeks gestation). The neonate's actual birth weight was used to validate the published foetal weight estimation models and modified sonographic birth weight estimation model was derived for our population by using linear regression. RESULTS: Modified sonographic birth weight estimation model for our population was derived by using foetal parameters. No significant difference (p-value > 0.05) of actual and predicted birth weight derived from Our regression model, Campbell and Woo models was noted, however least difference (p = 0.7) was identified between our predicted model (Mean difference 14 +/- 37.7 g). CONCLUSION: Our sonographic modified regression model of foetal weight estimation gave the least difference with actual neonatal birth weight and can be reliably used in our population. Hadlock1, Hadlock2 and Woo2 models are not appropriate in our setting or should be used carefully while predicting foetal weight in our population.